Halogenation of Complex Organic Compounds

ABSTRACT

The disclosure provides biocatalysts that halogenate complex chemical compounds in specific and predictable ways. Also disclosed are halogenated complex organic compounds. The disclosure further provides methods for the halogenation of complex chemical compounds and methods of inhibiting the contraction of smooth muscle in mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No. 17/228,211, filed Apr. 12, 2021, which is a Divisional of U.S. application Ser. No. 16/516,102, filed Jul. 18, 2019, which claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/700,152, filed Jul. 18, 2018, the disclosure of which is incorporated herein by reference in its entirety.

Incorporation by Reference of Material Submitted Electronically

The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “53295C_Seqlisting.XML”, which was created on May 8, 2023 and is 66,649 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under CA047135, CA070375 and R01 086374 awarded by the National Institutes of Health, and CHE-1205646 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD

The disclosure relates generally to the field of complex chemistry and more particularly to biocatalysis of compound halogenation.

BACKGROUND

The prevalence of halogenated natural products has led to significant advances in understanding various classes of halogenases involved in secondary metabolism. Most halogenases characterized thus far can be placed into three classes: haloperoxidases (heme-containing and vanadium-containing), non-heme Fe(II)α-ketoglutarate-dependent, and flavin-dependent enzymes. Haloperoxidases are generally nonselective and perform halogenation through a mechanism utilizing freely diffusing hypohalous acid. By contrast, Fe(II)α-ketoglutarate-dependent halogenases proceed through a radical mechanism, typically halogenating aliphatic, unactivated carbons.¹ Flavin-dependent halogenases (FDHs) also proceed through a hypohalous acid intermediate, with the reactive reagent captured by a lysine residue that appears to control the regioselectivity of halogenation on aromatic substrates.^(2,3) The FDH-derived hypohalous acid is generated through a reaction between the flavin C4a-peroxide adduct and the bound chloride ion. FDHs are thought to proceed through an electrophilic aromatic substitution (EAS) where the catalytic lysine residue provides the chloramine halogenating agent and a catalytic glutamate facilitates the reaction by deprotonating the positively charged intermediate generated during catalysis.²

The majority of previously characterized FDHs are of bacterial origin, with relatively few reported from eukaryotes,⁴⁻¹² and fewer still characterized biochemically.^(4-6,10-12) The bacterial FDHs have been found to catalyze reactions on both free,^(2,13-15) and carrier-protein-bound substrates,^(16,17) including precursor amino acids in natural product biosynthesis. The well-characterized eukaryotic FDHs Rdc24 and ChIA6 catalyze late-stage C—H functionalization reactions in the biosynthesis of halogenated metabolites. However, structural data for these two enzymes have not been reported, and it has remained unclear how they control site-selective halogenation on large, structurally complex substrates.

Malbrancheamide (compound 1) is a complex halogenated indole alkaloid produced by the terrestrial fungus Malbranchea aurantiaca RRC181318 and the marine sponge-derived fungus Malbranchea graminicola 086937A.¹⁹ The discovery of malbrancheamide was enabled by a search for calmodulin antagonists, and several studies have characterized its significant vasorelaxant effect.^(20,21) Along with malbrancheamide, a close structural relative, spiromalbramide, was isolated from M. graminicola. ¹⁹ The two strains are highly related, with 99% sequence identity overall, and their biosynthetic pathways for malbrancheamide are proposed to be identical (Scheme 1). Malbrancheamide (compound 1) belongs to a family of prenylated indole alkaloids formed through peptide coupling by a nonribosomal peptide synthetase (NRPS), addition of an isoprene unit by a prenyltransferase, and a proposed [4+2] Diels-Alder cycloaddition to form the characteristic bicyclo[2.2.2]diazaoctane ring of prem-albrancheamide (compound 2) (Scheme 1).²²⁻²⁶ Premalbrancheamide is then proposed to be dichlorinated through an iterative mechanism, but whether this halogenation is performed by one or two halogenases remained to be determined.²⁵ The chlorination of the indole ring differentiates this molecule from the rest of its class and significantly contributes to its biological activity.²¹

In earlier efforts to elucidate the malbrancheamide biosynthetic pathway, precursor incorporation studies were performed in M. aurantiaca. This led to the conclusion that premalbrancheamide (compound 2) is indeed incorporated into the monochlorinated malbrancheamide B (compound 3) and that both compounds are natural metabolites of M. aurantiaca. ²⁵ Previously, it had been proposed that there is a site-selective chlorination of the C9 position prior to functionalization of C8 for production of malbrancheamide (compound 1).²⁵ The isolation of both C8 (isomalbrancheamide B (compound 4)) and C9 (malbrancheamide B (compound 3)) monohalogenated metabolites from M. aurantiaca ²⁰ and from M. graminicola ¹⁹, however, conflicted with the proposed C9 selectivity hypothesis.

The ability to selectively halogenate C—H bonds in highly complex molecules through synthetic methods has posed a formidable challenge due to the abundance of chemically equivalent C—H bonds, and the inability to overcome inherent steric or electronic bias for reactivity.^(31,32) The large number of biologically active natural products that undergo late-stage functionalization by tailoring enzymes provides a unique opportunity to leverage the power of halogenating enzymes to perform difficult chemical transformations.

In view of the foregoing observations, a need continues to exist in the art for catalysts that modify complex compounds, for example the controlled halogenation of complex compounds such as indole alkaloids.

SUMMARY

The disclosure provides flavin-dependent halogenases (FDHs) that are amino acid variants of fungal MalA or MalA′ halogenases useful in specifically halogenating complex compounds such as indole alkaloids. These enzymes are useful in producing a variety of halogenated compounds in the fungal malbrancheamide pathway that have physiological effects in mammals such as inhibiting calmodulin and, thereby, modulating calcium ion signaling pathways involved in a diverse set of physiological pathways and, hence, a variety of diseases or disorders. Exemplary methods comprising administration of effective amounts of the compounds disclosed herein include inhibiting the contraction of smooth muscle in a mammal.

In one aspect, the disclosure provides a flavin-dependent halogenase (FDH) variant comprising one or two amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein the FDH variant is capable of catalyzing the halogenation of a complex organic compound. In some embodiments, the complex organic compound is an aromatic heterocyclic organic compound. In some embodiments, the aromatic heterocyclic organic compound comprises a bicyclo[2.2.2]diazaoctane ring. In some embodiments, the aromatic heterocyclic organic compound comprises indole. In some embodiments, the compound is an indole alkaloid. In some embodiments, the indole alkaloid is a prenylated indole alkaloid. In some embodiments, the FDH variant is derived from a fungal FDH. In some embodiments, the FDH variant is derived from a bacterial FDH. In some embodiments, the FDH variant is not derived from a fungal FDH or a bacterial FDH.

In some embodiments, the FDH variant is a MalA halogenase variant. In some embodiments, the FDH variant comprises an amino acid substitution variant of the sequence set forth in SEQ ID NO:2, wherein the substitution is S129Z, H253Z, S129Z/H253Z, D109Z, F489Z, S409Z, W265Z, W263Z, S82Z, S129Z/G131Z, G131Z, S129Z/I493Z, I493Z, S129Z/P85Z, or P85Z, wherein Z is no amino acid or any conventional amino acid except the wild-type amino acid at the indicated position. In the context of the disclosure, a substitution mutation or variation at the amino acid level is expressly defined as including the substitution of any naturally occurring conventional amino acid for a non-identical amino acid, or the substitution of no amino acid for a given amino acid, i.e., a single amino acid deletion. In some embodiments, the FDH variant comprises an amino acid substitution variant of the sequence set forth in SEQ ID NO:2, wherein the substitution is S129A, H253F, S129A/H253F, H253A, D109A, F489H, S409A, W265A, W263A, S82A, S129A/G131S, G131S, S129A/I493S, I493S, S129A/P85S, or P85S. In some embodiments, the MalA halogenase comprises the sequence set forth in SEQ ID NO:6, 8, 12, or 16. In some embodiments, the FDH variant is a MalA′ halogenase variant. In some embodiments, the FDH variant comprises an amino acid substitution variant of the sequence set forth in SEQ ID NO:4, wherein the substitution is E494Z or H253Z, wherein Z is no amino acid or any conventional amino acid except the wild-type amino acid at the indicated position. In some embodiments, the FDH variant comprises an amino acid substitution variant of the sequence set forth in SEQ ID NO:4, wherein the substitution is E494D, H253F, S129A, S129A/I493S, I493S, S129A/P85S, P85S, S129A/G131S, or G131S. In some embodiments, the MalA′ halogenase comprises the sequence set forth in SEQ ID NO:10, 14, or 18.

Another aspect of the disclosure is drawn to a polynucleotide encoding a FDH variant disclosed herein. In some embodiments, the encoded FDH variant is a MalA halogenase variant or a MalA′ halogenase variant. In some embodiments, the encoded FDH variant comprises a sequence set forth in SEQ ID NO:6, 8, 10, 12, 14, 16, or 18.

Yet another aspect of the disclosure is directed to a vector comprising the polynucleotide disclosed herein. A related aspect of the disclosure is a host cell comprising the polynucleotide disclosed herein or the vector disclosed herein.

Another aspect of the disclosure is a malbrancheamide D compound comprising formula I:

A related aspect of the disclosure is an isomalbrancheamide D compound comprising formula II:

A related aspect of the disclosure is a malbrancheamide E compound comprising formula III:

Still another aspect of the disclosure is a method of halogenating a complex organic compound comprising contacting a complex organic compound with a FDH halogenase variant disclosed herein and a halogen under conditions suitable for enzyme-catalyzed halogenation of the complex organic compound. In some embodiments, the complex organic compound is an aromatic heterocyclic organic compound. In some embodiments, the aromatic heterocyclic organic compound comprises a bicyclo[2.2.2]diazaoctane ring. In some embodiments, the aromatic heterocyclic organic compound comprises indole. In some embodiments, the compound is an indole alkaloid. In some embodiments, the indole alkaloid is a prenylated indole alkaloid. In some embodiments, the prenylated indole alkaloid is derived from a Malbranchea species. In some embodiments, the Malbranchea species is Malbranchea aurantiaca or Malbranchea graminicola. In some embodiments of the method, the prenylated indole alkaloid is premalbrancheamide, malbrancheamide B, isomalbrancheamide B, malbrancheamide C, or isomalbrancheamide C. In some embodiments, the halogenation step is a chlorination step. In some embodiments, the halogenation step is a bromination step.

Another aspect of the disclosure is drawn to a method of modulating a Ca²⁺ signaling pathway in a cell of a mammal or a cell in vitro comprising administering an effective amount of a halogenated complex organic compound to the mammal, the cell in vitro, or to the isolated enzyme or enzymes in vitro. Some embodiments implement a method of modulating a Ca²⁺ signaling pathway in a cell of a mammal comprising administering an effective amount of a halogenated complex organic compound to the mammal. In some embodiments, the Ca²⁺ signaling pathway is a Ca²⁺-calmodulin dependent pathway. In some embodiments, modulating the Ca²⁺ signaling pathway inhibits smooth muscle contraction. In some embodiments, the halogenated complex organic compound is malbrancheamide, malbrancheamide B, isomalbrancheamide B, malbrancheamide C, isomalbrancheamide C, malbrancheamide D, isomalbrancheamide D, or malbrancheamide E.

Some embodiments implement a method of modulating a Ca²⁺ signaling pathway in a cell, such as a mammalian cell, in vitro or in an isolated enzyme or enzymes in vitro comprising administering an effective amount of a halogenated complex organic compound to the cell in vitro or to the isolated enzyme or enzymes in vitro. Some embodiments provide a method of modulating a Ca²⁺ signaling pathway in a cell in vitro comprising administering an effective amount of a halogenated complex organic compound to the cell in vitro. In some embodiments, the Ca²⁺ signaling pathway is a Ca²⁺-calmodulin dependent pathway. In some embodiments, the Ca²⁺ signaling pathway being modulated in vitro participates in smooth muscle contraction in vivo. In some embodiments, the halogenated complex organic compound is malbrancheamide, malbrancheamide B, isomalbrancheamide B, malbrancheamide C, isomalbrancheamide C, malbrancheamide D, isomalbrancheamide D, or malbrancheamide E. Some embodiments implement a method of modulating a Ca²⁺ signaling pathway comprising administering an effective amount of a halogenated complex organic compound to an isolated enzyme or enzymes. In some embodiments, the Ca²⁺ signaling pathway is a Ca²⁺-calmodulin dependent pathway. In some embodiments, the Ca²⁺ signaling pathway being modulated in vitro participates in smooth muscle contraction in vivo. In some embodiments, the halogenated complex organic compound is malbrancheamide, malbrancheamide B, isomalbrancheamide B, malbrancheamide C, isomalbrancheamide C, malbrancheamide D, isomalbrancheamide D, or malbrancheamide E.

Other features and advantages of the disclosure will be better understood by reference to the following detailed description, including the drawing and the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1 . Scheme 1. Malbrancheamide biosynthetic pathway in M. aurantiaca and M. graminicola, where spiromalbramide is produced only in the latter.

FIG. 2 . Malbrancheamide biosynthetic gene clusters in M. aurantiaca and M. graminicola.

FIG. 3 . Malbrancheamide and related metabolites. M. aurantiaca isolates included compounds 1, 2, 3, and 4, while M. graminicola also produced compounds 5 and 6. Compounds 7 and 8 had not been previously described from either organism. See FIG. 1 for compound identities.

FIG. 4 . HPLC traces (240 nm) for the MalA in vitro reactions (A) chlorination of compound 2 (see FIG. 1 ) (ii) compared to standards from the fungal extract (i), (B) bromination of compound 2 (ii) compared to no enzyme control (NEC) (i), and (C) bromination of compound 4 (iii) compared to NEC (i) and bromination of compound 3 (iv) compared to NEC (ii). See FIG. 1 for compound identities.

FIG. 5 . gHMBCAD and gCOSY NMR correlations for the indole region used to determine the sites of halogenation of new malbrancheamide analogs.

FIG. 6 . MalA′ active site overlay of complexes with substrates 2 (yellow), 3 (pink), and 4 (cyan). See FIG. 1 for the identities of substrates, or compounds, 2, 3, and 4.

FIG. 7 . Active site of MalA′, revealing clear separation between the substrate and FAD binding pockets.

FIG. 8 . Percent conversion with mutants versus wild-type MalA in reactions with compound 2 to produce compounds 4 (green), 3 (blue), and 1 (red). MalA K108A and E494A/Q were inactive and MalA S82A was insoluble, and was not functionally analyzed. See FIG. 1 for compound identities.

FIG. 9 . Interactions between MalA′ Phe489 (orange) and substrates (A) premalbrancheamide (compound 2), (B) malbrancheamide B (compound 3), and (C) isomalbrancheamide B (compound 4). See FIG. 1 for compound identities.

FIG. 10 . Comparison of wild-type MalA′ (yellow) and MalA′ E494D (cyan) co-crystallized with compound 2. See FIG. 1 for compound identities.

FIG. 11 . Comparison of wild-type MalA′ (yellow) and MalA′ H253F (cyan) co-crystallized with compound 2. See FIG. 1 for compound identities.

FIG. 12 . a) Representative snapshot (at 40 ns) taken from the 500 ns MD simulation of MalA′ and substrate 2-bound complex, including the chloramine adduct at Lys108. b) Computed DFT reaction pathways for the three models: 1a-indole and methyl chloramine; 1b-indole, methyl chloramine and a water molecule coordinating at the C8 or C9 positions, respectively; 1c-indole, methyl chloramine, and MeOH coordinating to H—C8. c) DFT optimized geometries of some representative structures. Gibbs energies are given in kcal/mol, and distances in Å.

FIG. 13 . Scheme 2. Proposed mechanism for MalA catalysis.

FIG. 14 . Phylogenetic tree of characterized and uncharacterized (labeled with producing organism) FDHs. Putative fungal FDHs with high amino acid identity (greater than 50%) to the MalA cluster in the blue region are expected to contain the Zn²⁺-binding motif based on sequence alignments. The green region contains previously characterized bacterial FDHs (the exception is the eukaryotic ChIA) and the yellow region denotes a new subclass of putative FDHs from fungi. Phylogenetic tree prepared using Lasergene MegAlignPro (DNASTAR).⁴⁰

FIG. 15 . MalA variants display a range of site-selectivity on the natural substrate premalbrancheamide. The substrate position can be shifted to achieve site-selectivity.

FIG. 16 . Wild-type MalA (pink) reactions compared to mutants in black (A) MalA′ (MalA L276P/R428P), (B) MalA W263A, (C) MalA W265A, (D) MalA S409A, (E) MalA H253A, (F) MalA F489H. The reactions are composed of MalA or MalA variants combined with a nonspecific flavin reductase, excess flavin cofactor, NaCl as a source for chloride ion, and NADH cofactor. Analysis was performed by monitoring the absorbance at 240 nm by HPLC.

FIG. 17 . (A) Wild-type MalA reaction compared to (B) MalA D109A, and (C) MalA S129A. The reactions are composed of MalA or MalA variants combined with a nonspecific flavin reductase, excess flavin cofactor, NaCl as a source for chloride ion, and NADH cofactor. Analysis was performed by monitoring the absorbance at 240 nm by HPLC.

FIG. 18 . (A) Wild-type MalA reaction compared to (B) MalA S129A, (C) MalA H253F, and (D) MalA S129A/H253F. The reactions are composed of MalA or MalA variants combined with a nonspecific flavin reductase, excess flavin cofactor, NaCl as a source for chloride ion, and NADH cofactor. Analysis was performed by monitoring the absorbance at 240 nm by HPLC.

FIG. 19 . Chlorination of compounds 5 and 6 to produce compounds 8 and 7. Top (A) standard compound 5, (B) standard compound 8, (C) control compound 5, (D) chlorination reaction of compound 5. Bottom, (A) standard compound 6, (B) standard compound 7, (C) control compound 6, (D) chlorination reaction of compound 6, (E) chlorination reaction of compound 2 to produce compound 1 standard.

FIG. 20 . Bromination of compound 2 with MalA H253A and MalA H253F compared to wild-type MalA. (A) standard compound 2, (B) standard compound 5, (C) standard compound 6, (D) WT MalA bromination reaction with compound 2, (E) MalA H253A bromination reaction with compound 2, and (F) MalA H253A bromination reaction with compound 2.

FIG. 21 . DFT optimized reaction pathways for the C8 and C9 chlorination using three computational models: a) an indole ring and methyl chloramine; b) an indole ring, methyl chloramine and a water molecule closer to C8- or C9-H respectively; c) an indole ring, methyl chloramine and a methanol molecule as a Ser129 model. Relative Gibbs free energies (ΔG, in kcal/mol) are computed at the M06-2×/6-311+G(d,p)/CPCM(Diethylether)//M06-2x/6-31G(d)/CPCM(Diethylether) level. Bond lengths are in Å.

DETAILED DESCRIPTION

The fungal genus Malbranchea includes species such as M aurantiaca and M. graminicola that contain biosynthetic pathways for the production of complex halogenated compounds such a halogenated indole alkaloids that have useful physiological effects such as being useful in modulating Calcium ion signaling pathways in mammalian cells involved in many physiological functions and implicated in a variety of diseases and disorders. The disclosure provides variants of fungal halogenases such as the MalA halogenase of M. aurantiaca and the MalA′ halogenase of M. graminicola that catalyze the synthesis of complex halogenated compounds useful, e.g., in inhibiting smooth muscle contraction. The data provide a comprehensive characterization of the fungal halogenase variants and provide extensive characterizations of the structure and properties of the compounds used as substrates or produced by the halogenase variants, which generally fall into the malbrancheamide synthetic pathways of the Malbranchea fungal species.

The experiments disclosed herein were designed to identify and characterize the versatile halogenases involved in malbrancheamide biosynthesis and to demonstrate their potential as biocatalysts for halogenation of complex organic compounds, including various compounds found in the malbrancheamide pathway, such as premalbrancheamide (compound 2) and both mono- and di-halogenated malbrancheamide pathway compounds including malbrancheamide B (compound 3), isomalbrandheamide B (compound 4), malbrancheamide C, isomalbrancheamide C, malbrancheamide D, and isomalbrancheamide D (see FIGS. 1 and 3 ). The experiments probe the mechanism of the iterative late-stage halogenation of premalbrancheamide at two adjacent positions on the indole ring system. The isolation of both C8 (isomalbrancheamide B (compound 4)) and C9 (malbrancheamide B (compound 3)) monohalogenated metabolites from M. aurantiaca ²⁰ and from M. graminicola ¹⁹ conflicted with the proposed C9 selectivity hypothesis, providing further motivation to investigate the malbrancheamide halogenation process.

Genome sequencing and bioinformatic analyses of M. aurantiaca and M. graminicola led to the identification of MalA and MalA′, respectively. These two FDHs are 99% identical, differing by only two amino acids, and are proposed to catalyze dihalogenation as the last step in the malbrancheamide biosynthetic pathways of each organism (FIG. 2 ).²⁶ The late-stage halogenation of free substrate by a flavin-dependent halogenase from an NRPS-containing gene cluster is unusual. Halogenation typically occurs as the first step prior to activation of an amino acid in bacterial non-ribosomal peptide biosynthesis.¹ Of the previously characterized flavin-dependent halogenases, most act on subunit substrates such as single amino acids,^(2,13-15,27) or carrier protein-tethered small molecules.^(16,17,28,29) In terms of late-stage activity on a complex polycyclic substrate, the closest comparison to MalA is the cyanobacterial-derived WelO5 non-heme Fe(II)α-ketoglutarate-dependent halogenase, which acts on fischerindole and hapalindole alkaloids.³⁰ In addition to substrate scope analyses, halogen selectivity has also been explored in flavin-dependent halogenases, and the majority were found to catalyze both chlorination and bromination reactions. In precursor incorporation studies using high bromide salt concentrations in the marine fungal strain M. graminicola, bromination of premalbrancheamide (compound 2) led to the production of malbrancheamide C (5) and isomalbrancheamide C (compound 6) (FIG. 3 ).¹⁹

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. The two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains have been characterized. MalA and MalA′ catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogs were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA′ in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases, and provides new insights into a reaction mechanism that is expected to be unique.

The experimental results disclosed herein provide an example of a unique subclass of flavin-dependent halogenases that performs iterative late-stage halogenation of complex substrates independent of a carrier protein. MalA is encoded in a gene cluster containing a Non-Ribosomal Peptide Synthetase (NRPS), but the evidence provided herein demonstrates that this protein catalyzes effective late-stage functionalization on free substrates. The data lead to the expectation of a new mechanism, involving Ser129, for deprotonation of the positively charged Wheland intermediate in MalA/MalA′ halogenation (Scheme 2 as shown in FIG. 13 ). The hydrogen bond between Glu494 and the indole nitrogen is expected to increase the nucleophilicity of the aromatic ring. This facilitates the electrophilic aromatic substitution (EAS) reaction, producing the positively charged intermediate. A water molecule or serine side chain can then deprotonate the Wheland intermediate, leading to re-aromatization of the indole ring system (Scheme 2, FIG. 13 ).

As revealed in the following examples, two critical residues in the active site significantly contributed to substrate binding, Glu494 and Phe489. Glu494 hydrogen bonds with the indole nitrogen and Phe489 facilitates a favorable hydrophobic interaction with the aromatic ring of the indole. The activity of MalA F489H was significantly decreased relative to the wild-type MalA, especially for the second chlorination reaction. It is expected that the phenylalanine residue in the back of the active site aids in substrate binding, and maintains interactions with the monochlorinated products to facilitate a second halogenation reaction.

The Michaelis-Menten model kinetics displayed equal rates of monochlorination at both the C9 and C8 sites of compound 2, and equal rates for chlorination of compounds 3 and 4, leading to the conclusion that MalA is equally selective for both sites of the indole ring. Interestingly, the catalytic efficiency (k_(cat)/K_(m)) values for the second chlorination were twice those observed for the first chlorination, thus the initial chlorination is expected to prime the substrate for the second halogenation. This effect can be correlated with the structural data where the chlorine atom on the indole substrates interacts favorably with Phe489, potentially facilitating a better mode of binding. Additionally, the dichlorinated malbrancheamide (compound 1) did not bind in crystals, suggesting a faster dissociation rate for the dichlorinated than either of the monochlorinated products, which bound readily in MalA′ crystals.

In efforts elucidate the mechanism of selectivity in MalA, a histidine residue near the catalytic lysine was used to probe the active site region. MalA H253A displayed selectivity for chlorination at the C9 position of compound 2, while MalA H253F was selective for the C8 position of compound 2. MD simulations and DFT calculations demonstrated how key interactions between polar amino acid side chains (Ser129) or water molecules in the active site with the different C—H positions of the indole can control the selectivity of the chlorination reaction. This is accomplished by enhancing the nucleophilicity of the carbon atom during the EAS, but also by pre-organizing a base to carry out the final deprotonation step. An alanine substitution at His253 prevented the interaction of Ser129 and H—C8, leading to an overall apolar active site environment, favoring chlorination at C9. On the other hand, the C8 selectivity observed in MalA H253F can be explained by a more effective Ser129 interaction with H—C8. When investigating these mutants in the site-selectivity of the bromination reaction, the wild-type product profile was observed. Compared to HOCl, HOBr is a milder halogenating reagent, thus favoring the inherently more reactive C9 site of compound 2. These findings demonstrate that even small modulations to the active site region can lead to significant changes in the site-selectivity of the halogenation reaction.

The designation of MalA into a new class of flavin-dependent halogenases is exemplified not only by its reactivity but also by its unique structural motifs: a Zn²⁺-binding C-terminus and an expansive active site, able to accommodate complex substrates. The discovery of this Zn²⁺-binding motif provides a fingerprint for use in mining sequence data for MalA homologs in pursuit of biocatalysts for late-stage halogenation (FIG. 13 ). Investigation of MalA/MalA′ has provided new insights into the biocatalytic mechanism for iterative late-stage halogenation of complex substrates.

The following examples are presented by way of illustration and are not intended to limit the scope of the subject matter disclosed herein.

EXAMPLES Example 1 Materials and Methods

M. graminicola Genomic DNA Extraction and Sequencing

The filamentous fungal strain Malbranchea graminicola was cultivated on a static 100 mL potato dextrose broth (PBD) medium for 10 days at 26° C. The genomic DNA (gDNA) extraction and sequencing protocols are the same for that used in Li, et al.¹ and Solexa sequencing was used to determine genome sequences.

M. aurantiaca cDNA Preparation

Malbranchea aurantiaca was cultured for 15 days in PDB shaking at 160 rpm at 28° C. The Invitrogen Purelink RNA Mini Kit was used with the Plant and Fungal Tissue Processing protocol from the associated RNeasy Mini Handbook (2010) to isolate the RNA prior to treatment with DNase. Invitrogen Superscript first strand synthesis was used with the Protoscript M-MuLV First Strand cDNA Synthesis Kit and protocol to generate the cDNA. The malA coding region was amplified from the cDNA template by PCR using the primers below and the following PCR cycle: (1) 94° C. for 2 minutes, (2) 98° C. for 10 seconds, (3) 66.3° C. for 30 seconds, (4) 68° C. for 2 minutes, repeating steps 2-4 40 times.

Primers

(SEQ ID NO: 19) 5′-GAGAGCTAGCATGGCGCCGACACCAAAGTATACGT-3′ (SEQ ID NO: 20) 5′-CATTAAGCTTCTATGCAGCTGGCCTGGTAGGGGTT-3′ Cloning of malA-pMCSG7 and malA′— pMCSG7

The malA PCR product was inserted into the pMCSG7 vector by ligation independent cloning (LIC).² Escherichia coli XL1 Blue cells were transformed with malA-pMCSG7 for screening and plasmid maintenance. malA′— pMCSG7 was prepared though site-directed mutagenesis as described below. The HpaC flavin reductase (phaC plasmid) has been described.³

M. aurantiaca Growth and Extraction of Malbrancheamides

The isolation and purification procedure was adapted from Martinez-Luis, et al.⁴ Individual flasks of 75 mL potato dextrose broth were inoculated with 100 μL spore stock of M. aurantiaca and grown for three weeks, or until a white fungal mat was produced. Prior to the noticeably orange sporulation, the cultures were pulverized and extracted with dichloromethane. The crude extract was acid-base purified first with 1 M HCl, then neutralized with 2 M ammonium hydroxide to pH 9, and back extracted with dichloromethane. The extract was then purified by chiral HPLC on a Phenomenex Lux 5 μm Cellulose-3 250×10 mm column. The following HPLC time program was used for separation and purification of the malbrancheamide compounds: 50% acetonitrile for 18 minutes, gradient to 55% acetonitrile over 2 minutes, 55% acetonitrile for 2 minutes, gradient to 40% acetonitrile over 2 minutes, 40% acetonitrile for 5 minutes, at a flow rate of 4 mL/minute. The mobile phase consisted of water and acetonitrile. From a 1.5 L growth of M. aurantiaca, the following yields of the naturally occurring malbrancheamides were obtained: 1.6 mg/L premalbrancheamide (compound 2) (¹H-NMR, 400 MHz, CD₃OD, δ 1.24 (s, 3H), 1.34 (s, 3H), 1.42 (m, 1H), 1.85 (m, 3H), 1.94 (d, J=11.3 Hz, 1H), 1.99 (d, J=12.8 Hz, 1H), 2.14 (m, 2H), 2.21 (d, J=10.2 Hz, 1H), 2.78 (d, J=15.2 Hz, 1H), 2.89 (d, J=15.3 Hz, 1H), 3.01 (m, 1H), 3.42 (d, J=10.4 Hz, 1H), 7.02 (t, J=7.5 Hz, 1H), 7.07 (t, J=7.6 Hz, 1H), 7.25 (d, J=8.1 Hz, 1H), 7.35 (d, J=7.7 Hz, 1H), 2.6 mg/L isomalbrancheamide B (compound 4), 4.4 mg/L malbrancheamide B (compound 3), and 5.8 mg/L malbrancheamide (compound 1). The structures of the compounds disclosed herein were unequivocally established by NMR and MS studies in comparison to the previous isolation of these molecules. These materials enabled subsequent biochemical and structural studies of MalA and MalA′ as well as mutants or variants thereof, as disclosed herein.

MalA Expression and Purification

Expression of malA, malA′, and malA/A′ mutants. E. coli strain BL21 (DE3) was transformed with malA-pMCSG7 and the pGro7 chaperone plasmid set (GroEL/GroES) from Takara. Ampicillin (0.1 mg/mL), chloramphenicol (35 μg/mL), and L-arabinose (0.5 mg/mL) were added to 1 L of Terrific Broth (TB) media, which was then inoculated with the transformed E. coli cells. The 1 L cultures were grown at 37° C. until an OD₆₀₀ of 0.8-1.0 was reached, cooled at 20° C. for one hour, induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and expressed for 18 hours at 20° C.

Expression of malA′ for selenomethionyl MalA′. 450 mL selenomethionine medium (AthenaES) was supplemented with 25 mL TB media, and 150 μg/mL seleno-DL-methionine. Ampicillin (0.1 mg/mL), chloramphenicol (35 μg/mL), and L-arabinose (0.5 mg/mL) were added to the medium, which was then inoculated with the transformed E. coli cells. The cell cultures were grown at 37° C. until an OD₆₀₀ of 0.6 was reached, cooled at 20° C. for one hour, induced with 0.1 mM IPTG, and expressed for 18 hours at 20° C.

Protein purification for chlorination assays and large-scale reactions. The cell pellet from a 500 mL culture was re-suspended in 30 mL lysis buffer_(NaCl) (10% (v/v) glycerol, 500 mM NaCl, 20 mM imidazole pH 7, 20 mM HEPES pH 7). The cell suspension was supplemented with 50 μM flavin adenine dinucleotide (FAD) and cells were lysed with 5 mg lysozyme, 2 mg DNase, and 3 mM MgSO₄. Cell lysis was completed through sonication and cell waste was cleared through centrifugation under standard cell debris pelleting conditions (e.g., 39,200 rcf for minutes). The supernatant was filtered and MalA was purified through metal affinity chromatography on a 5 mL His-trap column (GE Healthcare) with a 10-column volume gradient of elution buffer_(NaCl) (10% glycerol, 500 mM NaCl, 30-560 mM imidazole pH 7, 20 mM HEPES pH 7). The protein was incubated on ice with 2 mM ATP and 50 μM FAD and further purified by size-exclusion chromatography on a Superdex S200 16/60 HiLoad column with storage buffer_(NaCl) (10% glycerol, 300 mM NaCl, 20 mM HEPES pH 7) to remove the chaperone proteins. 20 mg purified MalA were obtained per 1 L of cell culture.

Protein purification for bromination assays and large-scale reactions. A cell pellet from a 500 mL expression culture was re-suspended in 30 mL lysis buffer_(NaBr) (50 mM NaH₂PO₄, mM imidazole pH 7, 300 mM NaBr, 10% glycerol) and supplemented with 50 μM FAD. Cell lysis was accomplished through addition of 5 mg lysozyme, 2 mg DNase, and 3 mM MgSO₄ and sonication. Cell waste was cleared through centrifugation under standard cell debris pelleting conditions (e.g., 39,200 rcf for 25 minutes), and the protein was purified through batch binding with 10 mL Ni-NTA Superflow resin (Qiagen). The resin-bound protein was washed with wash buffer_(NaBr) (50 mM NaH₂PO₄, 20 mM imidazole pH 7, 300 mM NaBr, 10% glycerol) and the protein was eluted with elution buffer_(NaBr) (50 mM NaH₂PO₄, 250 mM imidazole pH 7, 100 mM NaBr, 10% glycerol, 0.2 mM TCEP). Bromide-bound MalA was exchanged into storage buffer_(NaBr) (50 mM NaH₂PO₄, 1 mM EDTA, 0.2 mM DTT, 10% glycerol, pH 7.3) on a PD-10 column (GE Healthcare).

Protein purification for crystallography. The initial steps of the purification were identical to those for the purification of MalA for chlorination assays. The His-tag was cleaved by TEV protease (1 mg protease/50 mg MalA) in an overnight dialysis with storage buffer_(NaCl), supplemented with 50 μM FAD and 2 mM DTT. Tag-free MalA was separated from TEV protease and any remaining Hiss-MalA by metal affinity chromatography, and purified by size exclusion chromatography with storage buffer_(NaCl). 10 mg of pure MalA were obtained per 1 L of cell culture.

MalA Biochemical Activity Assays

Biochemical activity assays were performed in a 100 μL volume with the following components: 18 μM MalA, 54 μM HpaC flavin reductase,³ 100 μM FAD, 50 mM NaCl, 250 μM substrate, 5 mM NADH, and brought to total volume with reaction buffer (same as storage buffer_(NaBr)). The chlorination reactions proceeded for 20 minutes and the bromination reactions proceeded overnight. The reactions were extracted with ethyl acetate (2004, in triplicate) and dried down under nitrogen gas. The dried extract was resuspended in LC/MS grade methanol to a concentration of around 10 μM for LC/MS analysis. High resolution mass spectrometry was performed using electrospray ionization on an Agilent quadrupole time-of-flight spectrometer (Q-TOF 6500 series). Biochemical activity was monitored via the following HPLC method using acetonitrile and water: 65% acetonitrile for 5 minutes, gradient over 10 minutes to 95% acetonitrile, 95% acetonitrile for 5 minutes, gradient over 2 minutes to 65% acetonitrile, 65% acetonitrile for 11 minutes to re-equilibrate with a flow rate of 0.3 mL/minute, monitoring at 240 nm on a Phenomenex Lux cellulose-3, cellulose Tris (4-methylbenzoate) 250×4.6 mm column.

Co-Crystal Structures of MalA′

Crystallization conditions. MalA from M. aurantiaca was recalcitrant to crystallization, but MalA′ from M. graminicola (generated by site-directed mutagenesis of malA: L276P/R428P) proved optimal for crystallization. The purified MalA′ was dialyzed overnight into a 20 mM HEPES pH 7 buffer with 200 mM NaCl or 300 mM NaCl to remove glycerol, and then supplemented with an equimolar quantity of FAD. Pre-incubation of MalA′ with an equimolar concentration of isomalbrancheamide B (compound 4) resulted in crystals with compound 4 bound in a lattice contact and not in the active site. For active site complexes with compounds 2, 3, and 4, MalA′ was pre-incubated with a four-fold molar excess of substrate. Crystals were grown by vapor diffusion from 1:2 mixtures of 5 mg/mL MalA′ pre-incubated with compound 2, 3 or 4 and a well solution containing 2 M (NH₄)₂SO₄, 0.2 M Li₂SO₄, 5 mM CdCl₂, and 0.1 M Bis-Tris pH 5.5. Crystals were cryoprotected in well solution augmented with 10% glycerol and flash-cooled in liquid nitrogen.

Data collection. Data were collected at GM/CA beamline 23ID-B at the Advanced Photon Source (APS) at Argonne National Laboratory. For the SeMet-MalA′ crystal, 180° of diffraction data were collected in inverse-beam geometry using 30° wedges. All data were processed using XDS.⁵ The SeMet MalA′ halogenase structure was solved by single-wavelength anomalous diffraction (SAD) using AutoSol in the Phenix suite to locate the Se sites, determine initial phases and perform density modification (figure of merit=0.236).⁶ AutoBuild in the Phenix suite was used to build an 82% complete starting model. The SeMet MalA model was used as a template in molecular replacement to solve the native MalA structure using Phaser in the Phenix suite. A progression of model building and refinement were carried out to complete the models using Coot and Phenix Refine with seven translation/libation/screw groups.⁷

Site-Directed Mutagenesis

MalA′ (MalA L276P/R428P). The site-directed mutagenesis (SDM) to prepare the L276P/R428P double substitution was performed sequentially starting with R428P. The reaction included 100 ng malA-pMCSG7 template, 100 ng each primer (forward_(L276P) and reverse_(L276P)) 5 μL 10× Pfu buffer, 0.5 μL dNTPs (250 μM each), and 1 μL PfuTurbo from Agilent in a total of 50 μL. The PCR cycle was (1) 95° C. for 30 seconds, (2) 95° C. for 30 seconds, (3) for 1 minute, (4) 68° C. for 8 minutes; steps 2-4 were repeated for 16 cycles. Dpnl digestion contained 0.5 μL 2 U/μL Dpnl and 25 μL PCR reaction solution for 2 hours at 37° C. and was performed prior to plasmid isolation by alkaline lysis (Purelink Quick Plasmid Miniprep Kit from Invitrogen) and sequencing to verify the presence of the mutant. The L276P substitution was prepared using single primer SDM with 100 ng malA R428P template, 0.2 μM primer, 250 μM dNTPs, 5 μL 10× Pfu buffer, 1 μL Pfu fusion polymerase in a total volume of 50 μL. The PCR time program was as follows: (1) 95° C. for 3 minutes, (2) 95° C. for 35 seconds, (3) 52° C. for 50 seconds, (4) 65° C. for 13 minutes, (5) 65° C. for 15 minutes; steps 2-4 were repeated for 30 cycles. Dpnl digestion and sequence analysis were performed in the same manner as described above.

Primers

R428P_(forward) (SEQ ID NO: 21) 5′-GCACAGCTTTCGCACCCAATTGTGGAGATTGGG-3′ R428P_(reverse) (SEQ ID NO: 22) 5′-CCCAATCTCCACAATTGGGTGCGAAAGCTGTGC-3′ L276P (SEQ ID NO: 23) 5′-CGTCTACCCTCTTGGGAAGGGAGCCCCATAGCGAACTTGATGGATA TGG-3′

Ma/A K108A. The malA K108A mutant was prepared using the Quikchange Lightning Site-Directed Mutagenesis Kit and protocol. The PCR time program used was (1) 95° C. for 2 minutes, (2) 95° C. for 20 seconds, (3) 55° C. for 30 seconds, (4) 65° C. for 6 minutes, (5) 65° C. for minutes; steps 2-4 were repeated for 30 cycles. The QCL Dpnl digest and transformation protocol were used with XL10-Gold Ultracompetent cells.

Primer

(SEQ ID NO: 24) 5′-GTAAAAGCACAGCCCATCCGCGAGTCCGAATAGTCGAAGG-3′

All other malA/malA′ mutants. The mutants were prepared using single primer SDM with 100 ng malA or malA′ template, 0.2 μM primer, 250 μM dNTPs, 5 μL 10× Pfu buffer, 1 μL Pfu fusion polymerase in a total volume of 50 μL. The PCR time program was as follows: (1) for 3 minutes, (2) 95° C. for 35 seconds, (3) X° C. (see below) for 50 seconds, (4) 65° C. for 13 minutes, (5) 65° C. for 15 minutes; steps 2-4 were repeated for 30 cycles. Dpnl digestion and sequence analysis were performed in the same manner as described above.

Primers

Mutant Primer X (° C.) S409A 5′-GGTTTCACCAACCCGCTCTATGCCCCGGGGATTAATGTTGG-3′ (SEQ ID NO: 25) 50.0 S82A 5′-CCTGGTTACAAGATTGGCGAGGCGACTCTACCTATCTTTTACACCTGG-3′ (SEQ 50.8 ID NO: 26) E494A 5′-GGCAGTTTTTCGCTGGCATAGCGCGATATTTGTCAGATGTTAACATTGAAACC-3′ 49.0 (SEQ ID NO: 27) E494Q 5′-GGCAGTTTTTCGCTGGCATACAGCGATATTTGTCAGATGTTAACATTGAAACC-3′ 49.0 (SEQ ID NO: 28) W263A 5′-CCACCTGTGTTTTCCGGAAGGTGCTGTCTGGGTTATTCGTCTACCCTCTTGGG-3′ 55.0 (SEQ ID NO: 29) W265A 5′-CCACCTGTGTTTTCCGGAAGGTTGGGTCGCGGTTATTCGTCTACCCTCTTGGG-3′ 55.0 (SEQ ID NO: 30) H253A 5′-CCCTTTGATCTCTATGAAGGTGATGCGACAAACCACCTGTGTTTTCC-3′ (SEQ ID 48.0 NO: 31) F489H 5′-CCCCAGGTGGCATGCCTCTGGCAGCATTTCGCTGGCATAGAGCG-3′ (SEQ ID 55.0 NO: 32) C613S/ 5′-CCGCCCAGATTGGAAAAAGTCTCACTCATCTGGTCTTCTGGGCACCG-3′ (SEQ ID 49.0 C616S NO: 33) C112S 5′-GGACTCAAGGATGGGCTGTCTTTTTACTTTCTTGATCGAGAGAACC-3′ (SEQ ID 49.6 NO: 34) C128S 5′-GGGGCAGTACACAGACTTCTCTAGTGTTGGGGCTCCAGGTTTGG-3′ (SEQ ID 53.7 NO: 35) E494D 5′-GGCAGTTTTTCGCTGGCATAGATCGATATTTGTCAGATGTTAACATTGAAACC-3′ 50.0 (SEQ ID NO: 36) H253F 5′-CCCTTTGATCTCTATGAAGGTGATTTTACAAACCACCTGTGTTTTCC-3′ (SEQ ID 48.0 NO: 37) S129A 5′-GGGGCAGTACACAGACTTCTGCGCGGTTGGGGCTCCAGGTTTGG-3′ (SEQ ID 55.0 NO: 38) D129A 5′-CCTTCGACTATTCGGACTCAAGGCGGGGCTGTGCTTTTACTTTCTTGATCG-3′ 50.0 (SEQ ID NO: 39)

MalA Large-Scale Reactions and Isolation of Products

Chlorination reaction conditions and extraction. Reactions were run in 1 mL aliquots with 90 μM MalA, 54 μM HpaC flavin reductase, 250 μM compound 2, 100 μM FAD, 50 mM NaCl, 5 mM NADH, and brought to total volume with reaction buffer (same as storage buffer_(NaBr)). Reactions were extracted after 20 minutes with 2 mL ethyl acetate in triplicate, dried under nitrogen gas, and re-suspended in methanol for HPLC purification. In a 5.1 mg reaction, 1.7 mg malbrancheamide B (compound 3), 1.3 mg isomalbrancheamide B (compound 4), and 1.2 mg malbrancheamide (compound 1) were isolated.

Bromination reaction conditions and extraction. Reactions were run in 1 mL aliquots with 40 μM MalA, 54 μM HpaC flavin reductase, 250 μM compound 2, 100 μM FAD, 50 mM NaBr, 5 mM NADH, and brought to total volume with reaction buffer (same as storage buffer NaBr). Reactions were extracted after 12 hours with 2 mL ethyl acetate in triplicate, dried under nitrogen gas, and resuspended in methanol for HPLC purification. In a 3.7 mg reaction with substrate (compound) 2, 0.9 mg malbrancheamide C (compound 5) and 0.7 mg isomalbrancheamide C (compound 6) were isolated. In a 2 mg reaction with compound 3, 480 μg compound 7 and 300 μg compound 1 were isolated. In a 2.3 mg reaction with compound 4, 1.8 mg compound 8 were isolated.

HPLC Purification. The malbrancheamide B (compound 3), isomalbrancheamide B (compound 4), and malbrancheamide (compound 1) products were purified using the same chiral HPLC method as for purification of the fungal extract. The malbrancheamide C (compound 5), isomalbrancheamide C (compound 6), malbrancheamide D (compound 7), and isomalbrancheamide D (compound 8) products were isolated using chiral HPLC with the previously mentioned semi-preparative cellulose column with the following HPLC time program: 70% acetonitrile for 14 minutes, gradient to 60% acetonitrile over 2 minutes at a flowrate of 4 mL/minute.

Michaelis-Menten Model Kinetics

Substrates malbrancheamide B (compound 3) and isomalbrancheamide B (compound 4) to product malbrancheamide (compound 1). Reactions were set up in a total volume of 250 μL with the following components: 1.1 μM MalA, 44 μM HpaC flavin reductase, 100 μM FAD, 50 mM NaCl, 3.6 mM NADH, and a variety of substrate concentrations ranging from 1 μM to 60 μM. Reactions were quenched with methanol by removing 50 μL at each time point (2, 5, 10, 15 minutes). Reactions were analyzed on a Schimadzu HPLC with the following LC time program: 40% acetonitrile for 1 minute, gradient over 6 minutes from 40-85% acetonitrile, 85% acetonitrile for 1 minute, gradient over 1 minute to 40% acetonitrile, re-equilibration to 40% acetonitrile for 3 minutes. The absorbance was measured at 240 nm and the mobile phase consisted of water and acetonitrile. A Phenomenex Lux cellulose-3, cellulose Tris (4-methylbenzoate) 250×4.6 mm column was used for separation. GraphPad Prism (Version 6.01) software was used to plot the initial velocities against the substrate concentration and to determine the kinetic constants k_(cat) and K_(m).

Substrate premalbrancheamide (compound 2) to products isomalbrancheamide B (compound 4) and malbrancheamide B (compound 3). Reactions were set up in a total volume of 250 μL with the following components: 1.8 μM MalA, 44 μM HpaC, 100 μM FAD, 50 mM NaCl, 3.6 mM NADH, and a variety of substrate concentrations ranging from 5 μM to 80 μM. Reactions were quenched with 100 μL methanol by removing 50 μL at each time point (2, 5, 7, 10 minutes). Reactions were analyzed on a Schimadzu HPLC with the following LC time program: 34% acetonitrile for 1 minute, gradient over 11 minutes to 62% acetonitrile, 62% acetonitrile for 30 seconds, gradient over 30 seconds to 34% acetonitrile, re-equilibration to 34% for 3 minutes. The absorbance was measured at 240 nm and the mobile phase consisted of water and acetonitrile. A Phenomenex Lux cellulose-3 Tris (4-methylbenzoate) 250×4.6 mm column was used for separation.

Computational Methods

DFT calculations. DFT calculations were performed using Gaussian 09 (Revision D.01).⁸ All geometries were optimized using M06-2X,⁹ within the CPCM polarizable conductor model (diethylether, ε=4),^(10,11) and the 6-31G(d) basis set. Single-point energies were calculated using the same DFT functional and solvation model, and the 6-311++G(d,p) basis set. The resulting energies were used to correct the gas-phase energies obtained from the M06-2X/6-31G(d) optimizations.¹² Enthalpies and entropies were calculated for 1 atm and 298.15 K. All stationary points were verified as minima or first-order saddle points by a vibrational frequency analysis. The use of a dielectric constant ε=4 has been proven to be a good and general model to account for electronic polarization and small backbone fluctuations in enzyme active sites and to have an estimation of the dielectric permittivity in the enzyme active site.^(13,14) Computed structures are illustrated with CYLView.¹⁵

Molecular Dynamics simulations. Molecular dynamics (MD) simulations were performed using the GPU code (pmemd)¹⁶ of the AMBER 16 package.¹⁷ Parameters for intermediate Cl—K and substrates were generated within the antechamber module using the general AMBER force field (gaft),¹⁸ with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the RESP model.¹⁹ The charges were calculated according to the Merz-Singh-Kollman scheme^(20,21) using the Gaussian 09 package.⁸ Each protein was immersed in a pre-equilibrated truncated cuboid box with a 10 Å buffer of TIP3P²² water molecules using the leap module, resulting in the addition of around 15,000 solvent molecules. The systems were neutralized by addition of explicit counter ions (Na⁺ and Cl⁻). All subsequent calculations were done using the widely tested Stony Brook modification of the Amber99 force field (ff99sb).²³ A two-stage geometry optimization approach was performed. The first stage minimizes the positions of solvent molecules and ions imposing positional restraints on the solute by a harmonic potential with a force constant of 500 kcal·mol⁻¹·Å⁻² and the second stage minimizes all the atoms in the simulation cell except those involved in the harmonic distance restraint. The systems were gently heated using six 50 ps steps, incrementing the temperature by 50 K for each step (0-300 K) under constant-volume and periodic-boundary conditions. Water molecules were treated with the SHAKE algorithm such that the angle between the hydrogen atoms was kept fixed. Long-range electrostatic effects were modeled using the particle-mesh-Ewald method.²⁴ An 8 Å cutoff was applied to Lennard-Jones and electrostatic interactions. Harmonic restraints of 30 kcal·mol⁻¹ were applied to the solute and the Andersen equilibration scheme was used to control and equalize the temperature. The time step was kept at 1 fs during the heating stages, allowing potential inhomogeneities to self-adjust. Each system was then equilibrated for 2 ns with a 2 fs time step at a constant volume. Production trajectories were then run for an additional 500 ns under the same simulation conditions.

REFERENCES FOR EXAMPLE 1 ONLY

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Example 2 Biochemical Activity of MalA

Purification of MalA by Ni-affinity chromatography and gel filtration provided pure protein for in vitro assays, and MalA was found to catalyze the iterative chlorination and bromination of the natural precursor premalbrancheamide (compound 2). In reactions with the monochlorinated compounds 3 and 4, MalA was also able to chlorinate and brominate these compounds to generate compounds 1, 7, 8, and 9 of which the latter three are novel indole alkaloids. The chlorination reaction of MalA was confirmed by co-elution with standards isolated from M. aurantiaca (FIG. 4 ). ¹H-NMR analysis confirmed the halogenation site on the indole ring and the high-resolution mass spectrometry data for all compounds were consistent with the expected masses and isotope peak patterns for the halogenated products.

The percent conversions of the halogenation reactions were determined by isolated yields. The chlorination of compound 2 to produce compounds 3, 4, and 1 in a 5 mg in vitro reaction showed 34%, 26%, and 24% conversion, respectively. The bromination of compound 2 in a 4 mg reaction generated 23% compound 5 and 18% compound 6 by isolated yield, but the calculated conversions based on standard curves displayed a C9 selectivity with an 8:1 ratio of 5 to 6. The methodology for separation of these monohalogenated intermediates by HPLC is well resolved compared to previous reports, thus the NMR data for the individual molecules significantly adds to the literature of these brominated indole alkaloids. MalA was also used as a biocatalyst for the generation of two novel malbrancheamide analogs, i.e., compounds 7 and 8 (FIGS. 4 and 5 ). The bromination of compound 3 in a 2 mg reaction produced 24% compound 7 and 15% compound 1 as a side product. The bromination of compound 4 in a 2.3 mg reaction produced 78% compound 8. The structural assignments of compounds 7, 8, and 9 were confirmed through extensive 1D and 2D NMR analyses. The structures were confirmed using key gHMBCAD correlations (FIG. 5 ) where a significant downfield shift was observed for the chlorinated carbon as opposed to the brominated carbon. The positions of the halogens on the indole ring were confirmed by the two singlet peaks observed in the ¹H-NMR aromatic region of each spectrum. Moreover, compounds 5 and 6 can also be chlorinated to produce compounds 8 and 7, respectively.

Example 3 Kinetic Characterization of MalA

Michaelis-Menten kinetic parameters were determined for the natural chlorination reactions of MalA. They revealed that the enzyme has similar k_(cat) and K_(m) values for both the initial and second chlorination reactions. The k_(cat) from compound 2 to compound 3, compound 2 to compound 4, compound 3 to compound 1, and compound 4 to compound 1 were 0.08, 0.09, 0.12, and 0.12 min⁻¹, respectively, which are comparable values to those of FDH PrnA (0.10 min⁻¹).² The K_(m) values for compound 2 to compound 3, compound 2 to compound 4, compound 3 to compound 1, and compound 4 to compound 1 were 7.0, 7.5, 4.4, and 4.0 μM. The catalytic efficiencies were calculated for each of the four reactions resulting in the k_(cat)/K_(m) values of 11.5, 12.0, 27.3, and 29.7 min−1 mM−1, respectively. These catalytic efficiencies are fairly high compared to those of the eukaryotic FDH Rdc2, which are 2.93 min−1 mM−1 for the initial chlorination reaction and 0.11 min−1 mM−1 for the second chlorination reaction.⁴

Example 4 Structural Characterization of the Substrate Complexes of MalA′

To further elucidate the unique functionality of MalA/MalA′ reactivity at two sites, the co-crystal structures of a MalA′ in complex with premalbrancheamide (compound 2), malbrancheamide B (compound 3), and isomalbrancheamide B (compound 4) were determined. MalA and MalA′ are 99% identical, differing at only two amino acid positions (Leu276 and Arg428 in MalA; Pro276 and Pro428 in MalA′), and have comparable catalytic activities, but only MalA′ was amenable to crystallization. To verify that MalA′ was a viable substitute for MalA, the activities of the two were compared, and it was determined that MalA′ was essentially identical to MalA under the reaction conditions tested. The structures of the ternary complexes with FAD, chloride ion, and each of the three substrates (compounds 2, 3, and 4) were determined at 2.36 Å, 2.09 Å, and 2.04 Å, respectively. MalA′ has a similar overall structure to bacterial FDHs, with the addition of a few unique motifs including a Zn²⁺-binding C-terminus and a large active site capable of accommodating the structurally complex substrates. The natural substrates, compounds 2, 3, and 4, have a similar binding mode in the MalA′ active site (FIG. 6 ). Specific interactions include a hydrogen bond of the indole nitrogen to Glu494 and a Cl-π interaction of compounds 3 and 4 with Phe489 (FIGS. 6 and 9 ). The roles of amino acids in the active site were analyzed through site-directed mutagenesis (FIGS. 7 and 8 ), and Lys108 was determined to be necessary for catalytic activity.

Trp263 and Trp265 form a characteristic flavin-binding motif proposed to aid in cofactor binding (FIG. 7 ). While MalA W265A showed a drastic decrease in activity, MalA W263A showed a more modest decrease in product formation (60% production of compound 4, 50% production of compound 3, and 5% production of compound 1) relative to wild-type MalA. A residue key to binding the substrate (Phe489) was substituted with histidine to ascertain its significance in the activity of MalA and was found to decrease the activity as well. Phe489 is analogous to the phenylalanine whose mutagenesis altered the site-selectivity in the FDH PrnA.³³

In an effort to probe the mechanism of MalA, Glu494 was substituted with a variety of other residues including alanine, glutamine, and aspartate. While E494A and E494Q inactivated MalA, E494D maintained slight activity. Glu494 forms a hydrogen bond with the proton of the indole nitrogen, facilitating binding of the substrate. The substitution of aspartate at this position shifted the substrate away from the catalytic lysine, thus decreasing the activity (FIGS. 8 and 10 ).

Initial efforts to probe the mechanism of site-selectivity in MalA included substitution of His253 with alanine, phenylalanine, and other amino acids. MalA H253A was selective for producing the C9-chlorinated compound 3, while MalA H253F displayed selectivity for producing the C8-chlorinated compound 4. Co-crystal structures of MalA′ H253A in complex with compounds 2 and 3 revealed no evident changes in the protein that would lead to the observed site-selectivity. On the other hand, the co-crystal structure of MalA′ H253F in complex with compound 2 revealed a shift in S129, a residue near the indole ring of the substrate (FIG. 11 ). When S129 was substituted with alanine, the C8 selectivity of MalA H253F was abolished, leading to the conclusion that Ser129 is involved in the selectivity induced by the Phe substitution at position 253 (FIG. 8 ). Interestingly, the MalA H253A and MalA H253F mutants did not display the same site-selectivity profile for the bromination reaction. Instead, the same product profile as the wild-type MalA was found (see FIG. 20 ).

The structures of MalA′ also revealed a unique zinc site with coordination by four cysteine residues (Cys597, Cys600, Cys613, Cys616) near the C-terminus. The Zn²⁺ ion was identified using anomalous scattering experiments with diffraction data recorded at X-ray energies bracketing the zinc K-edge (9.6586 keV). Anomalous difference density was present only in the map using data from the energy above the edge. A double mutant MalA C613S/C616S was prepared and the protein was insoluble, thus no biochemical activity assays were performed in the absence of Zn²⁺.

Example 5 Molecular Dynamics Simulations and QM Models

Molecular dynamics (MD) simulations were performed to gain further insight into the structure and activity of the protein, starting from the MalA′ crystal structures in their apo and substrate bound forms. In the latter case, the Lys108 chloramine intermediate has been considered integral to the mechanism discussed below.

The analysis of the MD trajectories revealed the structural role played by the Zn²⁺ counterion in the protein structure. Residues within the Zn²⁺ binding region (597-616) exhibited a low root-mean-square fluctuation (RMSF) compared to the very flexible adjacent region (621-646). These simulations indicate that the flexible region acts as a substrate channel lid, having two main open/closed conformations that were explored during the 500 ns MD simulations in both the apo and substrate-bound states.

From the apo state trajectories, the pK_(a) of the Lys108 and Glu494 side chains was estimated. Glu494 has a relatively high pK_(a) of about 6.0-7.5, while Lys108 has an estimated pK_(a) of 7.2-8.3.

The analysis of possible polar interactions between the substrate and the enzyme active site showed that, although the backbone carbonyls of Gly131 and Ala132 could potentially interact with the substrate amide nitrogen, these interactions are not as important as the Glu494—H(N-indole) hydrogen bond. The latter corresponds to the main and stronger interaction between the substrate and protein active site residues, and it is observed with all bound substrates (compounds 2, 3 and 4) during the entirety of the MD trajectory simulations. The basicity of Glu494 can thus enhance the hydrogen bonding between the carboxyl side chain and the indole ring of the substrate, positioning it to effectively interact with the catalytic Lys108 residue.

MD simulations including the proposed active chloramine Cl-Lys108 species showed that when substrate 2 (compound 2) is bound into the active site and the channel lid is closed, the CI atom is placed very close to the C8/C9 positions of the substrate, due in part to the positioning of the substrate by the Glu494 residue (see FIG. 12 a ). When the lid is in its open conformation, the substrate binding is slightly displaced, although still H-bonding with Glu494, but then the Cl-Lys108 side chain conformation changes to place the CI atom closer to the FAD cofactor. This indicates that the protein conformational change between the substrate bound open/closed states also controls the positioning of Cl-Lys108 active species, which can explore two main conformations to bring the CI atom from the flavin cofactor to the substrate. This observation supports the expectation that Cl-Lys108 is the actual chlorinating species. When Cl-Lys108 is in this catalytically competent pose, the distances (Cl—C) and angles (Cl—C—H) measured for both C8 and C9 positions are very similar, indicating that Cl-Lys108 is preorganized to chlorinate both positions.

MD simulations also revealed a key interaction between Cl-Lys108 and the backbone carbonyl of the neighboring Asp109 residue, effectively positioning Cl-Lys108 towards a catalytically competent arrangement (FIG. 12 a ). The CI atom from Lys108 chloramine active species can only be placed close to the C8/C9 positions when this H-bond is present. The essential role of the Asp109 was further explored by mutagenesis experiments, and the D109A mutant had very low activity (FIG. 8 ). MD simulations on this particular mutant showed that the Ala109 backbone carbonyl is pointing towards a different position than in the original Asp109, thus eliminating a key hydrogen bond interaction. This is caused by the different conformation of the amino acid side chain, which is pointing outside the protein cavity and exposed to the solvent in Asp109, while in Ala109 it is displaced. Indeed, in the 500 ns simulations for the D109A mutant, Cl-Lys108 never explores any conformation in which the CI atom approaches the C8/C9 positions to perform chlorination.

MD simulations on MalA′ Cl-Lys108 with bound substrate 2 (compound 2) highlighted the arrangement of the Ser129 side chain with respect to H—C8 in compound 2. The distance between Ser129 Oy and H—C8 is particularly short (between 2.5-3.5 Å) when the substrate is in an appropriate orientation for the electrophilic aromatic substitution. This interaction was not observed along the MD trajectory of MalA′ H253 Å, but was prominent in the MalA′ H253F trajectory. This interaction is quite important since Ser129 is one of the few polar residues in a very hydrophobic region of the active site pocket. Along the MD trajectories, the solvation shell estimated for the C8 and C9 positions of compound 2 showed a more apolar environment for wild-type MalA′ and MalA′ H253A (i.e., fewer surrounding water molecules) than for MalA′ H253F.

Based on the experimental evidence and computational modeling of the enzyme active site, a mechanism for the MalA halogenase was developed (FIG. 12 , Scheme 2) involving the formation of a Lys108-chloramine intermediate active species, which then interacts with C8 or C9 to carry out an electrophilic aromatic substitution (EAS), with generation of a Wheland intermediate (W) before a final deprotonation step (Scheme 2). This deprotonation can be effected by a water molecule acting as a base, or in the case of C8 by the well-positioned Ser129. To gain insight into the proposed reaction mechanism, density functional theory (DFT) calculations were conducted, using three different computational models (see Example 1 for computational details). The first model considers only an indole ring and a protonated methyl chloramine as the active species (1a); the second adds a water molecule close to H—C8 or H—C9 positions (1b); and the third model includes a methanol molecule near H—C8 position to mimic Ser129 (1c). Calculations show that the intrinsic rate-limiting step of the reaction is the initial chlorination, while the deprotonation step occurs slightly faster. A water molecule or methanol interacting with the C8/C9 protons accelerates the chlorination steps because hydrogen bonding enhances the nucleophilicity of these carbons. The computed reaction barrier for C8-chlorination (TS1a-C8) was decreased from 6.4 to 5.5 kcal/mol by a coordinating H₂O (TS1b-C8), and further decreased to 3.5 kcal/mol when methanol coordinates to the H—C8 (TS1c-C8). On the other hand, the computed barrier for C9 chlorination (TS1a-C9) decreased from 7.0 to 5.2 kcal/mol by a coordinating H₂O molecule at H-C9 (TS1b-C9), and to 4.9 kcal/mol when methanol interacted with H—C8 (TS1c-C9), as shown in FIG. 12 . This highlights the role of Ser129 in directing the selectivity towards the formation of the C8 chlorinated product.

The Wa-C9 Wheland intermediate is 1.3 kcal/mol more stable than Wa-C8, but they become almost isoenergetic when coordinating water molecules are considered (Wb-C8 and Wb-C9). An apolar environment favors the formation of the C9 chlorinated product 3 (compound 3). Finally, once the Wheland intermediates are formed, re-aromatization by deprotonation occurs rapidly. The computed deprotonation barriers for the two positions are 4.1 and 3.5 kcal/mol for C8 (TS2b-C8) and C9 (TS2b-C9), respectively, when a water molecule acts as the base, and 0.6 kcal/mol for C8 when methanol acts as a base (TS2c-C8).

The DFT optimized structures for the reactant complexes and transition states are highly similar. The catalytically competent arrangement of Cl-Lys108 near C8 and C9 was found in the MD simulations (represented in FIGS. 12 a, 12 c ). Taking together the QM models, MD simulations, and the pre-organization of the Cl-Lys108 versus the substrate previously described, the reaction mechanism is expected to involve a Cl-Lys108 intermediate, which is the most plausible mechanism for the MalA flavin-dependent halogenase.

Example 6 Development of MalA Halogenase as a Biocatalyst for Late-Stage C—H Functionalization

The malbrancheamides are complex hexacyclic fungal indole alkaloids with biological activity as calmodulin antagonists, and the halogenation of the indole ring significantly contributes to the potency of the molecules. MalA has been characterized as an iterative late-stage halogenase that provides the halogen moieties to produce brominated and chlorinated malbrancheamide analogs. Experimental investigation into the mechanism of halogenation has identified a serine residue within the active site as pivotal to catalysis of halogenation. This knowledge base has been used to engineer a range of MalA variants for selective halogenation on the natural substrate premalbrancheamide. Structural and computational analyses of the mutants has aided in determining a mechanism for modulating the selectivity of the chlorination reaction. The substrate scope of the MalA-catalyzed reaction has been analyzed by screening 1,000 computationally predicted substrates. The experimental work disclosed herein has led to the identification of MalA variants, i.e., the C9-selective mutants MalA G131S and S129A/I493S, and the C8-selective mutant MalA S129 Å/P85S. See FIG. 15 . Crystal structures of the wild-type enzyme compared to variants have aided the visualization of how these mutations change the binding pocket and provide insight into the accommodation of unnatural substrates.

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All publications and patents mentioned in the application are herein incorporated by reference in their entireties or in relevant part, as would be apparent from context. Various modifications and variations of the disclosed subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for making or using the disclosed subject matter that are obvious to those skilled in the relevant field(s) are intended to be within the scope of the following claims. 

1-36. (canceled)
 37. A method of modulating a Ca²⁺ signaling pathway in a cell of a mammal comprising administering an effective amount of a halogenated complex organic compound to the mammal.
 38. The method of claim 37 wherein the Ca²⁺ signaling pathway is a Ca²⁺-calmodulin dependent pathway.
 39. The method of claim 37 wherein modulating the Ca²⁺ signaling pathway inhibits smooth muscle contraction.
 40. The method of claim 37 wherein the halogenated complex organic compound is malbrancheamide, malbrancheamide B, isomalbrancheamide B, malbrancheamide C, isomalbrancheamide C, malbrancheamide D, isomalbrancheamide D, or malbrancheamide 41-44. (canceled) 